Mechanisms determining the expansion of CD94^lowCD56^dimNK cell subset induced by MM plasma cells. (A) Frequency of CD94^lowCD56^dim NK cells among total NK cells from HDs upon 24-hour coculture with or without allogeneic MM cells isolated from the BM of 4 distinct patients or with the indicated tumor cell lines. E/T ratio in the culture = 1:1. (B) In vitro proliferation of NK cells from HDs cultured for 24 hours in the absence (medium) or presence of primary MM cells, U266B1 and K562 cell lines. The Ki67 expression on NK cells is shown in relation to CD94 molecule distribution. Summary of data from 3 independent experiments is shown as mean ± SEM. (C) Proliferation of the 3 indicated NK cell subsets following coculture with primary MM cells. (D) NK cells from HDs were cultured with U266B1 cells for 24 hours in the presence of blocking mAbs for the indicated activating receptors or isotype-matched control mAbs (IgM ctrl). Histograms represent δ (Δ) values ± SEM of percentage increase of CD94^lowCD56^dim NK cells, calculated as the percentage of CD94^lowCD56^dim NK cells cultured with U266B1 subtracted the percentage of CD94^lowCD56^dim NK cells cultured without target cells. Bars represent mean ± SEM from 6 independent experiments. (E) CD56^neg and CD56⁺ MM were sorted from patient’s BM (left dot plot) and cocultured for 24 hours with HD NK cells (right dot plots). One representative experiment of 3 with similar results is shown. (F) NK cells from HDs were cultured for 24 hours with MM cells isolated from patients’ BM in the presence of blocking mAb for CD56/NCAM-1 and then analyzed for the expression of CD94 by flow cytometry. Histograms show representative expression of CD94 in cultured NK cells in the presence of anti-CD56/NCAM-1 blocking mAb for or isotype-matched control mAb (IgM ctrl). Bars represent δ (Δ) values ± SEM of the increase in the percentage of CD94^lowCD56^dim NK cells calculated as the percentage of CD94^lowCD56^dim NK cells cultured with MM cells subtracted the percentage of CD94^lowCD56^dim NK cells cultured without a target. Data shown are from 6 independent experiments. (G) In vitro proliferation of HD NK cells cultured with MM cells in the presence of anti-CD56/NCAM-1 blocking antibody. Numbers adjacent to gates indicate the frequency of Ki67⁺ NK cells. Bars represent data from 4 independent experiments shown as mean ± SEM of Ki67⁺ cells. (H) Proliferation of HDs NK cells cocultured (24 hours) with either K562 or K562 transfected with the cDNA coding for CD56 protein. A representative experiment and mean of data ± SEM from 3 independent experiments are shown. **P < .01, ***P < .001; Student t test. n.s., not significant.